U.S. patent number 5,803,664 [Application Number 08/768,585] was granted by the patent office on 1998-09-08 for process for remediating soil.
This patent grant is currently assigned to Canon Kabushiki Kaisha, Raito Kogyo, Co, Ltd.. Invention is credited to Masatoshi Iio, Takeshi Imamura, Yuji Kawabata, Shinya Kozaki, Yuri Senshu, Michiyo Suzuki, Yoshiyuki Touge, Tetsuya Yano.
United States Patent |
5,803,664 |
Kawabata , et al. |
September 8, 1998 |
**Please see images for:
( Certificate of Correction ) ** |
Process for remediating soil
Abstract
A process for remedying polluted soil which comprises the steps
of injecting a liquid agent containing a microorganism into the
polluted soil, and injecting a gas into a region wherein the water
content of the soil became 0.6 or more times its saturation water
content after the injection of the liquid agent. Using this method
bioremediation of contaminated soil can be economically and
efficiently carried out.
Inventors: |
Kawabata; Yuji (Isehara,
JP), Yano; Tetsuya (Isehara, JP), Touge;
Yoshiyuki (Sagamihara, JP), Kozaki; Shinya
(Tokyo, JP), Imamura; Takeshi (Chigasaki,
JP), Iio; Masatoshi (Funabashi, JP),
Suzuki; Michiyo (Yotsukaido, JP), Senshu; Yuri
(Kashiwa, JP) |
Assignee: |
Canon Kabushiki Kaisha (Tokyo,
JP)
Raito Kogyo, Co, Ltd. (Tokyo, JP)
|
Family
ID: |
26555372 |
Appl.
No.: |
08/768,585 |
Filed: |
December 18, 1996 |
Foreign Application Priority Data
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Oct 25, 1899 [JP] |
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8-284212 |
Dec 19, 1995 [JP] |
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7-330428 |
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Current U.S.
Class: |
405/128.5;
166/246; 210/611; 210/747.7; 435/262.5 |
Current CPC
Class: |
B09C
1/10 (20130101); B09C 2101/00 (20130101) |
Current International
Class: |
B09C
1/10 (20060101); B09B 001/10 () |
Field of
Search: |
;166/246
;210/610,611,747 ;405/128 ;435/262.5 ;588/205,249 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0370409 |
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May 1990 |
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EP |
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0412472 |
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Feb 1991 |
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EP |
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3601979 |
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Jul 1987 |
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DE |
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1203194 |
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Jan 1986 |
|
SU |
|
Primary Examiner: Suchfield; George A.
Attorney, Agent or Firm: Fitzpatrick, Cella, Harper &
Scinto
Claims
What is claimed is:
1. A process for remediating a soil contaminated with a pollutant
by using a microorganism, comprising the steps of:
injecting a liquid containing a microorganism capable of degrading
the pollutant into a predetermined site of the soil region to be
remedied; and
injecting gas into the predetermined site where the liquid agent is
injected, wherein the gas injection step is conducted when a water
content of the site is 0.6 or more times a saturation water content
of the soil.
2. A process according to claim 1, wherein the pollutant is
phenol.
3. A process according to claim 1, wherein the pollutant is a
chlorinated organic aliphatic hydrocarbon.
4. A process according to claim 1, wherein the pollutant is
trichloroethylene.
5. A process according to claim 1, wherein the microorganism is
strain J1 (FERM BP-5102).
6. A process according to claim 1, wherein the microorganism is
strain JM1 (FERM BP-5352).
7. A process according to claim 1, wherein the liquid contains
dissolved gas.
8. A process according to claim 1, wherein the gas contains at
least one gas selected from the group consisting of air, oxygen,
nitrogen, carbon dioxide and methane.
9. A process according to claim 1, wherein the liquid further
contains nutrient for said microorganism.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for remediating polluted
soil.
2. Related Background Art
A large amount of chemical compounds or chemical products have been
produced with the recent rapid progress in technology and science.
Many of them do not originally exist in nature, thus they scarcely
decompose of themselves or decomposed naturally, and are slowly
accumulated in the environment and contaminate the nature. In
particular, the land, where the human beings live, is most easily
affected by artificial contamination, and since the environmental
water is circulating among the land, hydrosphere, and atmosphere,
the environmental pollution in the land is a serious problem which
might be expanded to the global level. Well-known examples of soil
(land) contaminants include organic compounds such as gasoline,
organochloric compounds such as PCB, teratogenetic agrichemicals
such as dioxin, and radioactive compounds. Particularly, fuel such
as gasoline has been stored widely and in a large amount in a gas
station's underground tank and the like, and the leakage of the
fuel into soil due to the deterioration or damage of the tank has
become a serious social problem. Organochloric compounds such as
trichloroethylene and tetrachloroethylene were once extensively
used for washing precision parts as well as for dry cleaning, and
large scale contamination of soil and underground water due to the
leakage of the organochloric compounds has been gradually revealed.
Since such organochloric chemicals are teratogenic and carcinogenic
to adversely affect the biosphere, purification of the polluted
soil or ground water is now an issue to be solved immediately in
addition to the isolation of the pollution source.
Methods to purify the soil contaminated by these contaminants
include a method in which the contaminated soil is dug out and
subjected to heat treatment, a method in which the contaminant is
extracted from the contaminated soil by vacuum extraction, or a
method using microorganisms which have such capabilities that
decompose these contaminants. By the heat treatment method, the
contaminants can be almost completely removed from the soil,
however, it requires digging of the soil, thus the purification of
the soil which is under buildings is hard to be carried out, and
the cost required for digging and heat-treatment are huge thus it
is difficult to apply this method to purify the contaminated soil
in a large area. The vacuum extraction process is an inexpensive
and simple purification process for volatile compounds, however,
the removal efficiency for an organochloric compound of some ppm or
less is low, and the purification thereof requires such a length of
time that is measured in years. On the other hand, the purification
method utilizing microorganisms do not require digging of the
contaminated soil, thus the soil under buildings can be purified
and by the use of microorganisms having high decomposing
activities, the contaminants can be decomposed and eliminated in a
short time, and it has been catching attention as an economical and
efficient soil purification method.
U.S. Pat. No. 5,133,625 describes a method in which the injection
pressure, flow rate and temperature are measured by using an
extendable injection pipe to control the injection pressure,
thereby the concentration of microorganisms and that of nutrients
in the soil, to carry out purification of soils efficiently. U.S.
Pat. Nos. 4,442,895 and 5,032,042 disclose a method of effective in
situ microbial remediation of polluted soil, where cracks are
formed in the soil using a liquid or a gas injected into the soil
with pressure from an injection well. U.S. Pat. No. 5,111,883
discloses a method for injecting chemicals vertically or
horizontally into a limited region of the soil by setting the
relative position of the injection and extraction wells.
It has been considered that the injection of a
pollutant-decomposing microorganism, nutrients, an inducer, oxygen,
and other chemicals into the soil is essential for the microbial
remediation of polluted soil. However, according to the
conventional injection methods, an extremely large amount should be
injected to remedy a wide area, since the liquid agent is injected
from the injection element to fill the soil void. Such a process
increases the processing period, labor and material costs,
resulting in increased remediation expenses. Differing from
chemicals, microorganisms can spontaneously grow and multiply when
certain growth conditions such as nutrient are satisfied. If a
liquid agent containing the microorganism and nutrient can be
injected in an amount as small as possible into a wide area of soil
and the microorganism can grow in the soil to decompose pollutants,
the purification expenses is considerably decreased. However, when
the necessary amount of the microorganism and nutrient is injected
into a wide area after dilution, the processing period and labor
required for injection do not decrease. Further, such a method that
the liquid agent will fill most of the soil void may cause soil
fluidization and soften the ground with a high possibility, it
cannot be applied to the soil under heavy structures. Moreover, the
liquid agent injected into the soil penetrates into the deeper
layers and diffuses into underground streams. Therefore, mobile
microorganisms and nutrients will not remain within the desired
area and lost. Thus, reinjection is required, making it difficult
to remedy soil at a low cost. Further, the runoff of the
microorganism and nutrient may cause secondary environmental
pollution. Consequently, in microbial soil purification, it is
required a method for injecting using a small amount of the agent
into a wide area of soil without filling all the void (pore space)
of the soil.
SUMMARY OF THE INVENTION
The present invention has been made in view of the above-mentioned
problems of the conventional techniques, and its objective is to
provide a soil remediation method in which a treating liquid agent
is distributed over a wide area by injecting a reduced quantity of
the liquid agent into the soil.
According to the present invention, there is provided a process for
remedying a soil contaminated with a pollutant by using a
microorganism, comprising the steps of:
injecting a liquid containing a microorganism capable of degrading
the pollutant into a predetermined site of the soil region to be
remedied; and
injecting gas into the predetermined site where the liquid agent is
injected, wherein the gas injection step is conducted when a water
content of the site is 0.6 or more times a saturation water content
of the soil.
The present invention is based on the finding that a small amount
of a liquid agent containing a microorganism and nutrients can be
distributed in a large area of the soil by injecting the agent from
an injection element into the soil, followed by injection of a gas
when the water content of the soil becomes 0.6 or more times as
much as its saturation water content due to the injection of the
liquid agent. It is also based on a finding that the injection
treatment can be carried out more effectively by repeatedly
injecting a liquid agent and a gas in turn where the gas is
injected when the water content becomes 0.6 or more times as much
as its saturation water content after the liquid injection.
In soil hardening technology which has no relation with the
microbial soil remediation, a treatment process has been known in
which a gas and a liquid agent for soil hardening are injected into
soil alternately. For example, SU No.1203194A describes a process
in which voids are produced in the soil around an injection port by
injecting a pressurized gas, and a chemical solution is infiltrated
into the soil by the gas pressure so that the soil is
press-hardened. Also, the jet grout technique is a known technique
for improving the soil ground by injecting very high pressure
water, compressed air and a hardening agent into the soil, in which
the soil is fractured by the high pressure-energy of water and air,
and a part of the soil is evacuated to the ground surface, while a
hardening agent is mixed into the soil for soil hardening. However,
these known techniques disclose nothing about the technical concept
of the present invention, that is, the injected liquid is carried
by the gas.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic illustration of one example of an apparatus
used for injecting a liquid agent in the present invention and FIG.
1B is an enlarged view of an end of an injection pipe of the
apparatus.
FIG. 2 is a graph showing the number of microorganism at the
sampling points in Example 1.
FIG. 3 is a graph showing the water content at the sampling points
in Example 1.
FIG. 4 is a graph showing the number of microorganism at the
sampling points in Example 2.
FIG. 5 is a graph showing the water content at the sampling points
in Example 2.
FIG. 6 is a graph showing the number of microorganism at the
sampling points in Comparative Example 1.
FIG. 7 is a graph showing the water content at the sampling points
in Comparative Example 1.
FIG. 8 is a graph showing the number of microorganism at the
sampling points in Comparative Example 2.
FIG. 9 is a graph showing the water content at the sampling points
in Comparative Example 2.
FIG. 10 is a graph showing the number of microorganism at the
sampling points in Example 3.
FIG. 11 is a graph showing the water content at the sampling points
in Example 3.
FIG. 12 is a graph showing the number of microorganism at the
sampling points in Example 4.
FIG. 13 is a graph showing the water content at the sampling points
in Example 4.
FIG. 14 is a graph showing the number of microorganism at the
sampling points in Comparative Example 3.
FIG. 15 is a graph showing the water content at the sampling points
in Comparative Example 3.
FIG. 16 is a graph showing the number of microorganism at the
sampling points in Comparative Example 4.
FIG. 17 is a graph showing the water content at the sampling points
in Comparative Example 4.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
When a liquid agent containing microorganisms and nutrients and the
like is injected under pressure into soil, the area wherein the
solution can be injected is mostly decided by the water permeation
coefficient of the soil and the amount of the solution injected.
For example, in the case of sand layer having a large water
permeation coefficient, the injected liquid agent spreads almost
spherically from the injection point filling the voids of the soil,
finally in a form of sphere the size of which is decided by the
amount of the injected solution and the porosity of the soil, while
a part of the solution falls down spontaneously due to its own
weight. In the case of loam layer having a smaller water permeation
coefficient, the injected liquid agent spreads in a vein-like form
along the weaker soil structure. In both cases, the injected liquid
agent fills the voids in the soil and migrates, thus a large amount
of the liquid agent is required, if the liquid agent is injected
and distributed in the soil of a large area. In order to carry out
the soil remediation economically and efficiently, it is preferable
to inject and distribute a liquid agent of minimal amount in an
extensive area of the soil, therefore it is preferable to migrate
the injected liquid agent in the soil without diluting the
solution.
Such migration of the liquid agent can be attained by injecting a
gas into a region of the soil where the water content of the soil
is maintained at a certain level due to the injected liquid agent,
illustratively the water content of 0.6 or more times and not more
than 1.0 times its saturation water content. The materials to be
contained in the liquid agent include, for example, a microorganism
which can decompose a chemical substance, a growth agent used for
microbial growth, an activity-supporting agent required for
decomposition activity expression, a survival agent required for
stable microbial inhabitation, an infiltration agent for easy
infiltration of the above materials, a channel forming agent for
enhancing infiltration of the liquid agent into the soil, and an
indicator for monitoring the infiltration of the liquid agent into
the soil. These materials may be used alone or in combination in
the liquid agent to be injected.
Examples of microorganisms which can decompose chemical pollutants
include those of genera Saccharomyces, Hansenula, Candida,
Micrococcus, Staphylococcus, Streptococcus, Leuconostoc,
Lactobacillus, Corynebacterium, Arthrobacter, Bacillus,
Clostridium, Neisseria, Escherichia, Enterobacter, Serratia,
Achromobacter, Alcaligenes, Flavobacterium, Acetobacter,
Nitrosomonas, Nitrobacter, Thiobacillus, Gluconobacter,
Pseudomonas, Xanthomonas, and Vibrio.
The growth agent means a nutrient for the microorganism, using
which the microorganism grows to decompose the chemical substance
in soil. As a useful growth agent, there are bouillon, M9, Malt
Extract, MY, or a selective medium for nitrifying bacteria. When
the degrading enzyme is constitutively expressed in the
microorganism, the activity-supporting agent is not necessary. When
the expression of the enzyme requires a specific inducer, such an
inducer must be added as the activity-supporting agent. Examples of
inducers include methane for methane-oxidizing bacteria; toluene,
phenol and cresol for aromatic-assimilating bacteria; ammonium
salts for nitrifying bacteria. Decomposing enzymes can also be
directly used for remedying polluted soil. In such a case, an
energy source or minerals required for the enzyme activity must be
added as the activity-supporting agent.
The survival agent provides a habitat for the useful microorganism,
preventing predation by other microorganisms and small animals, or
diffusion into ground water. Any microorganism-carriers used for
bioreactors in medical or food industries and waste water treatment
can be used as a survival agent. Examples of survival agent include
particulate carriers, e.g. porous glass, ceramics, metal oxides,
activated charcoal, kaolinite, bentonite, zeolite, silica gel,
alumina and anthracite; gel carriers, e.g. starch, agar, chitin,
chitosan, polyvinyl alcohol, alginic acid, polyacrylamide,
carrageenan, agarose, and gelatin; polymer resins and ion exchange
resins, e.g. cellulose, glutaraldehyde, polyacrylic acid and
urethane polymers. Further, natural and synthetic polymer
compounds, e.g. cellulose products such as cotton and hemp, pulp
paper, polymeric acetate which is a modified natural product, and
polyester and polyurethane cloths can be also used in the present
invention. Compost is a useful material which acts as both a growth
agent and a survival agent. Examples of compost materials include
straw, sawdust, rice bran, bagasse, and crab and lobster
shells.
Examples of channel forming agents include surfactants, e.g. sodium
dodecyl sulfate and TRITON-X, an ethoxylated alkylphenol sold by
Rohm & Haas; and foaming agents, e.g. sodium hydrogen carbonate
and ammonium carbonate. It is preferred that the indicator readily
dissolves or disperses in the liquid agent and readily detected at
the migration point when it migrates with the liquid agent.
Examples of indicators include water-soluble pigments which changes
the color of the soil, and organic acids and salts for changing the
electroconductivity of the soil.
Examples of a gas to be injected include air, oxygen, carbon
dioxide, nitrogen, hydrogen, helium, neon, argon, carbon monoxide,
methane, nitrogen monoxide, nitrogen dioxide, and sulphur dioxide.
By injecting such a gas into a part of the soil where a liquid
agent containing a microorganism and nutrient has been injected and
the soil becomes to have the water content of 0.6 or more times its
saturation water content, the liquid agent remaining in the voids
of the soil is pushed out and migrated further away from the
injecting element. For example, when 1 liter of a liquid agent is
injected into a fine sand layer of which water content is around
0.5 times its saturation water content, the water content of the
soil in a sphere of 8 cm radius from the liquid agent injection
port, becomes 0.8-1 times its saturation water content. If no gas
is injected, the liquid agent migrates downwards with time by its
own weight, and in about 2 to 3 hours, the water content of the
soil in the sphere becomes less than 0.6 times its saturation water
content. Therefore, according to the present invention, the
injection of a gas is preferably carried out immediately after the
injection of the liquid agent, illustratively at latest within 3
hours, more specifically within 2 hours, though it depends on the
soil condition. When the region in the soil into which the liquid
agent is to be injected or diffused has already a high water
content prior to the injection, the injected liquid agent will be
diluted. Thus, the water content of the soil where the liquid agent
is to be injected is preferably 0.5 or less times, preferably 0.45
or less times its saturation water content. When a liquid agent is
injected into and diffused through a region of the soil of a high
water content, the water content of the soil is preferably lowered
by, for example, injecting a gas prior to the injection of the
liquid agent. This preliminary gas injection also secures the voids
in the soil for injection of the liquid agent. By repeating a
process of injecting a liquid agent and a gas in this order, where
the latter is carried out while the water content of the soil is
higher than the predetermined level, the liquid agent can be
distributed in a soil region where originally the infiltration of
the liquid agent is not easy, so that the microorganism and
nutrient contained in the liquid agent can be more uniformly
distributed in the end. Furthermore, by sequentially changing the
concentration of the liquid agent component or that of the gas
component during the injection process, the concentration of the
components can be varied in the soil. Also, in this injection
process, by changing the components of the liquid agent or the gas,
the injected components can be distributed in the soil in a
localized fashion. By using such an injection method, the
concentration distribution of the injected components in the soil
can be changed and the decomposition activity of the microorganism
can be controlled from outside.
According to the method of injecting a liquid agent into soil
followed by a gas injection carried out while the water content of
the soil is higher than the predetermined level, the injected
solution is migrated and distributed in the extensive soil region
without filling almost all the voids in the soil, in other words,
soil voids filled with gas are remained. That means, the injection
treatment can be carried out without increasing the water content
of the soil to its saturation water content, thus the natural
dropping of the liquid agent by its own weight does not occur
easily and the outflow of the components such as the microbial
nutrient is small and so the secondary contamination caused
therefrom. When a gas containing a component necessary for the
growth and proliferation of the microorganism such as oxygen or
methane is used for injection, gas supply for the microorganisms
can be simultaneously carried out in addition to the injection and
distribution of the microorganisms and the nutrient.
One example of the liquid agent injection apparatus according to
the present invention is shown in FIG. 1A. The injection apparatus
comprises a liquid agent tank 1 to store the liquid agent, a liquid
agent pressure pump 2, a gas tank 3 to store the gas to be
injected, a pressure pump 4 by which the gas is sent under pressure
into the soil, and an injection pipe 5. In order to drive the
injected liquid agent further into the soil, The liquid agent is
injected into the soil by running the pump 2 for a predetermined
length of time, and then under the conditions that the water
content of the soil has become 0.6 or more times its saturation
water content due to the injection of the liquid agent, the pump 4
is operated for a predetermined length of time so that the gas is
injected to drive the liquid agent away. At this time, the amount
of the liquid agent to be injected and the amount of the gas to be
injected are controlled by the operation time of pump 2 and that of
pump 4 according to the area of the soil region to be treated, the
concentration of the contaminants, the microbial capacity of
decomposing the contaminant and the like. When the liquid agent
injection process and the gas injection process are carried out
repeatedly, each pump are operated repeatedly. When the
concentration or the components of the liquid agent or the gas is
changed, it is carried out in tank 1 and tank 3. By using a pump
suitable for introducing both gas and liquid, the liquid agent and
the gas can be injected into the soil by one pump. As an injection
pipe 5, a single pipe having an injection opening at the tip or on
the side can be utilized. When an injection operation is carried
out repeatedly changing the injection depth, it is useful to
combine a Manchette pipe 7 having a rubber sleeve 6 with a sleeve
pipe 9 having packers 8 as shown in FIG. 1B. That means, the sleeve
pipe 9 is moved up and down and set at a desired position, then the
upper and lower packers 8 are expanded, and the liquid agent is
sent under pressure through the sleeve pipe 9 to the part between
the upper and lower packers 8, and infiltrated into the soil under
pressure through the rubber sleeve 6. Then the air is injected
through the sleeve pipe 9 to drive the liquid agent further in the
soil. This process can be repeatedly carried out at different
injection depths.
As explained above, a small amount of the liquid agent can be
distributed in the soil of a large area according to each
embodiment of the present invention, and microbial purification of
the soil can be carried out economically and efficiently.
The present invention will be further illustrated with the
following examples but those examples are not to be construed to
limit the present invention.
EXAMPLES
Example 1
Migration of Microbial Cells and Change of Water Content in Column
with Injection of Liquid Agent Followed by Immediate Air
Injection
In a column of 5.0 cm in inner diameter and 100 cm long, a 5
cm-deep gravel layer was provided at the bottom of the column and
2900 g of fine sand was packed thereupon. The saturation water
content of the fine sand was 23% and the water content at the time
was 10%. On the packed fine sand, another gravel layer of 5 cm deep
was provided and the column was used in experiments. The depth of
the packed fine sand layer was 90 cm. As a microorganism, strain JI
(National Institute of Bioscience and Human-Technology, Agency of
Industrial Science and Technology: FERM BP-5102) was used. It was
cultured overnight in an M9 medium supplemented with 0.1% yeast
extract. The number of the microorganism of the overnight culture,
determined by the number of colonies formed on an agar medium, was
5.times.10.sup.8 cells/ml. The overnight culture was diluted 500
times with pure water, and the dilution of 1.times.10.sup.6
cells/ml was injected into the column as a liquid agent from the
bottom. The injection of the liquid agent was carried out using a
peristaltic pump at the pumping speed for injection of 50 ml/min.
The injection of the liquid agent was stopped when the liquid agent
reached 45 cm from the bottom of the sand layer, immediately
followed by injection of air under a pressure of 1 kg/cm.sup.2. Air
injection was continued until the water front in the sand was
observed to reach the upper end of the sand layer. The water
content of the soil in the lower 50 cm of the column immediately
after the injection of the liquid agent was about 22-23%, which was
determined using another similar column. The water content was
determined by weighing samples before and after drying at
120.degree. C. overnight.
Immediately after the air injection, the fine sand layer was taken
out of the column and the migration of the microorganism and the
change of the water content were determined. The sampling of the
sand layer was carried out at a total of 9 points, ranging from 5
cm from the bottom of the sand layer, to 85 cm from the bottom of
the sand layer, with an interval of 10 cm. The number of the
microorganism at each sampling point was determined as follows: 20
ml of pure water was added to 20 g of the sample, and vortexed for
30 seconds, followed by appropriate dilution. The dilution was
plated on an agar medium to count the number of the colonies
formed. The water content was determined as follows: each sample
was weighed and dried at 120.degree. C. overnight and from the
weight before and after drying the water content was calculated.
The number of the microorganism at each sampling point is shown in
FIG. 2 and the water content is shown in FIG. 3. These results show
that the microorganism and water were migrated efficiently in the
soil by sending air under pressure through an area where the water
content is 0.6 or more times its saturation water content. The
results also show that the localized increase of the water content
in the column can be controlled so that the water content can be
leveled in the entire column.
Example 2
Migration of Microbial Cells and Change of Water Content in Column
with Repeated Injection of Liquid Agent Followed by Immediate Air
Injection
In a column of 5.0 cm in inner diameter and 130 cm long, a 5
cm-deep gravel layer was provided at the bottom of the column and
3867 g of fine sand was packed thereupon. The saturation water
content of the fine sand was 23% and the water content at the time
was 10%. On the packed fine sand, another gravel layer of 5 cm deep
was provided and the column was used in experiments. The depth of
the packed fine sand layer was 120 cm. As a microorganism, strain
JI (FERM BP-5102) was used. It was cultured overnight in an M9
medium supplemented with 0.1% yeast extract. The number of the
microorganism of the overnight culture, determined by the number of
colonies formed on an agar medium, was 5.times.10.sup.8 cells/ml.
The overnight culture was diluted 500 times with pure water, and
the dilution of 1.times.10.sup.6 cells/ml was injected into the
column as a liquid agent from the bottom. The injection of the
liquid agent was carried out using a peristaltic pump at the
pumping speed for injection of 50 ml/min. The injection of the
liquid agent was stopped when the liquid agent reached 30 cm from
the bottom of the sand layer, and immediately followed by injection
of the air under a pressure of 1 kg/cm.sup.2. Air injection was
continued until the water front in the sand was observed to reach
60 cm from the bottom of the sand layer. Then the liquid agent was
injected again at the same pumping speed as in the first injection,
and the injection was stopped when the liquid agent reached 60 cm
from the bottom of the sand layer, then air was injected under the
pressure of 1 kg/cm.sup.2 again, so that the water front in the
fine sand was observed to be pushed up to 90 cm from the bottom of
the sand layer. The third injection of the liquid agent and air
under pressure was repeated in the same manner as in the previous
injection and the water front in the fine sand was pushed up to 120
cm from the bottom of the sand layer. The water content before each
air injection step was measured using three separately prepared
sand packed columns, and they were all 22-23%.
After the injection of air, the fine sand layer was taken out from
the column and the migration of the microorganism and change of the
water content were determined. Sampling of the sand layer was
carried out at a total of 12 points, starting from a point of 5 cm
from the bottom of the sand layer to a point of 115 cm from the
bottom of the sand layer, with an interval of 10 cm. The number of
the microorganism at each sampling point was obtained as follows:
20 ml of pure water was added to 20 g of the sample, and vortexed
for 30 seconds, followed by appropriate dilution. The dilution was
plated on an agar medium to count the number of the colonies
formed. The water content was determined as follows: each sample
was weighed and dried at 120.degree. C. overnight and from the
weight before and after drying the water content was calculated.
The number of the microorganism at each sampling point is shown in
FIG. 4 and the water content is shown in FIG. 5. These results show
that the microorganism and water were migrated efficiently in the
soil by sending air under pressure through an area where the water
content is 0.6 or more times its saturation water content. The
results also show that the localized increase of the water content
in the column can be controlled so that the water content can be
leveled in the entire column.
Comparative Example 1
Migration of Microbial Cells and Change of Water Content in Column
with Injection of Liquid Agent Alone
A procedure was carried out in the same manner as in Example 1
except that the injection of the liquid agent was continued until
the water front in the sand was observed to reach the upper end of
the column, and except that air injection was omitted. Then the
migration of microorganism and water content in the fine sand in
the column was observed in the same manner as in Example 1.
The number of the microorganism and the water content at each
sampling point are shown in FIG. 6 and FIG. 7 respectively. These
results show that the injection of the liquid agent alone only
increases the water content of the soil and does not allow the
microorganism to migrate efficiently.
Comparative Example 2
A process similar to that of Example 1 was carried out except that
the air injection was carried out 24 hours after the injection of
the liquid agent, to observe the migration of microorganism and
change of water content in the column. At that moment, almost all
the water in the column had been evacuated from the column, and the
water content at the bottom of the fine sand layer was about 0.5
times its saturation water content. The migration of the
microorganism in the fine sand layer and the change of the water
content after the injection of air are shown in FIG. 8 and FIG. 9
respectively.
Example 3
Migration of Microbial Cells and Change of Water Content in Pot
with Injection of Liquid Agent Followed by Immediate Air
Injection
In a stainless pot having an inner diameter of 32 cm, a length of
30 cm, a gravel layer of 5 cm deep and 30 kg of fine sand were
placed in this order to be used for experiments, where the
saturation water content and the water content of the fine sand was
23% and 10% respectively, and the height of the sand layer was 25
cm. As a microorganism, strain JI (FERM BP-5102) was used. It was
cultured overnight in an M9 medium supplemented with 0.1% yeast
extract. The number of the microorganism of the overnight culture,
determined by the number of colonies formed on an agar medium, was
5.times.10.sup.8 cells/ml. The overnight culture was diluted 500
times with pure water, and the dilution of 1.times.10.sup.6
cells/ml was injected into the pot as a liquid agent. The injection
of the liquid agent was carried out using a peristaltic pump in the
center of the pot, at the depth of 8 cm, and the pumping speed for
injection was 200 ml/min. The liquid agent was injected for 5
minutes, then without delay, about 40 l air was injected under a
pressure of 1 kg/cm.sup.2 for 3 minutes through the same injection
port. The water content of the soil before the air injection was
measured at 6 points within a sphere of a radius of 8 cm from the
injection port, using a separately prepared sand packed pot, and
they were about 22-23%.
Immediately after the air injection, the fine sand layer was taken
out from the pot to determine the migration of the microorganism
and the change of water content. Sampling of the sand layer was
carried out at a total of 12 points, i.e. at radii of 6 cm, 9 cm
and 12 cm from the center of the pot, each in the depth of 2 cm, 8
cm, 14 cm and 20 cm from the surface of the soil. The number of the
microorganism at each sampling point was obtained as follows: 20 ml
of pure water was added to 20 g of the sample, and vortexed for 30
seconds, followed by appropriate dilution. The dilution was plated
on an agar medium to count the number of the colonies formed. The
water content was determined as follows: each sample was weighed
and dried at 120.degree. C. overnight and from the weight before
and after drying the water content was calculated.
The number of the microorganism at each sampling point is shown in
FIG. 10 and the water content is shown in FIG. 11. These results
show that the microorganism and water were migrated efficiently in
the soil by sending air under pressure through an area where the
water content is 0.6 or more times its saturation water content.
The results also show that the localized increase of the water
content in the radius directions of the same depth in the pot can
be controlled so that the water content can be leveled at the same
depth.
Example 4
Migration of Microbial Cells and Change of Water Content in Pot
with Repeated Injection of Liquid Agent Followed by Immediate Air
Injection
A pot packed with fine sand and a liquid agent were prepared in the
same manner as that used in Example 3. The injection of the liquid
agent was carried out using a peristaltic pump at the center of the
pot at a depth of 8 cm, and the pumping speed for injection was 200
ml/min. The liquid agent was injected for 3 minutes, then about 40
l air was injected under a pressure of 1 kg/cm.sup.2 for 3 minutes
without delay from the same injection port. Immediately after the
injection of the air, the liquid agent and then air were injected
again in the same manner as in the first injection. The process was
repeated two more times. The water content immediately after each
injection of the liquid was measured by using separately prepared
sand packed pots were 22-23%.
The migration of the microorganisms and the change of water content
in the fine sand layer after injection of air were obtained in the
same manner as in Example 3. The number of the microorganism and
water content at each sampling point are shown in FIG. 12 and FIG.
13 respectively. These results show that the microorganism and
water were migrated efficiently in the soil by repeating the liquid
injection each followed by the air injection under pressure through
an area where the water content is 0.6 or more times its saturation
water content. The results also show that the localized increase of
the water content in the pot at the same depth can be controlled so
that the water content in the radius direction can be leveled.
Comparative Example 3
A process similar to that of Example 3 was carried out except that
the air injection into the soil was carried out 24 hours after the
liquid injection, to observe the migration of microorganism and the
change of water content in the pot. The water content measured
before the air injection at 6 points in a sphere of 8 cm radius
from the injection port were around 0.5 times its saturation water
content at maximum.
The migration of the microorganism in the fine sand layer and the
change of the water content after the air injection are shown in
FIG. 14 and FIG. 15 respectively.
Comparative Example 4
Migration of Microbial Cells and Change of Water Content in Pot
with Injection of Liquid Agent Alone
A procedure similar to that of Example 3 was carried out except
that injection of air was not carried out to observe the migration
of microbial cells and water content in the pot. The number of the
microorganism and the water content at each sampling point are
shown in FIG. 16 and FIG. 17 respectively. These results show that
the injection of the liquid agent alone only achieves uneven water
content in the soil and does not allow the efficient migration of
the microbial cells.
Example 5
Decomposition of Phenol in Column with Liquid Agent Injection
Followed by Immediate Air Injection
Columns packed with fine sand were prepared in the same manner as
in Example 1. Phenol was added to the sand to a concentration of
about 10 ppm for experiments. As a microorganism, strain JI was
used. It was cultured overnight in an M9 medium supplemented with
0.1% yeast extract. The number of the microorganism of the
overnight culture, determined by the number of colonies formed on
an agar medium, was 5.times.10.sup.8 cells/ml. The overnight
culture was diluted 500 times with pure water, and the dilution of
1.times.10.sup.6 cells/ml was injected into the column as a liquid
agent from the bottom. The injection of the liquid agent was
carried out using a peristaltic pump at the pumping speed for
injection of 50 ml/min. The injection of the liquid agent was
stopped when the liquid agent reached 45 cm from the bottom of the
sand layer, and immediately followed by injection of the air under
a pressure of 1 kg/cm.sup.2. Air injection was continued until the
water front in the sand was observed to reach the upper end of the
sand layer. The water content of the soil immediately after the
liquid injection measured in the same manner as in Example 1 was
22-23%.
The above-mentioned injection procedure was carried out in two
columns; the fine sand layer was taken out from one of the columns
immediately after the injection of the air, while the other column
was sealed tightly with a Teflon seal immediately after the air
injection, and after 5 days the fine sand was taken out from this
column for phenol concentration determination. The sampling of the
sand layer was carried out at 9 points, i.e. 5 cm from the bottom
of the sand layer to 85 cm from the bottom with an interval of 10
cm, and the phenol concentration at each sampling point was
measured according to JIS method (JISK 0102-1993, 28.1). The phenol
concentration at each sampling point is shown in Table 1. These
results show that the microorganism and water were migrated
efficiently in the soil and phenol in the soil was decomposed
efficiently by the process where the liquid injection was followed
by air injection under pressure into the region of which water
content is 0.6 or more times its saturation water content.
TABLE 1 ______________________________________ Phenol concentration
(ppm) in the column immediately after and 5 days after injection of
air Distance from the bottom of the sand layer (cm) 5 15 25 35 45
55 65 75 85 ______________________________________ Immediately 10 9
10 11 11 9 8 9 10 after injection 5 days after 0 0 1 0 1 0 0 0 0
injection ______________________________________
Comparative Example 5
Decomposition of Phenol in Column with Injection of Liquid Agent
Alone
Into a column of fine sand polluted with phenol, the liquid agent
was injected in the same manner as in Example 5.
After the injection of the liquid agent, the column was tightly
sealed with a Teflon seal and the fine sand layer was taken out
from the column after 5 days to determine the phenol concentration.
The sampling of the sand layer was carried out at 9 points 5 cm
from the bottom of the sand layer to 85 cm from the bottom at an
interval of 10 cm, and the phenol concentration at each sampling
point was measured according to JIS method (JISK0102-1993,28.1).
The phenol concentration at each sampling point is shown in Table
2. These results show that the injection of the liquid agent alone
does not allow the efficient migration of the microbial cells or
the efficient decomposition of phenol in the soil.
TABLE 2 ______________________________________ Phenol concentration
(ppm) in the column into which only the liquid agent was injected
Distance from the bottom (cm) of the sand layer 5 15 25 35 45 55 65
75 85 ______________________________________ 5 days after 0 0 0 1 3
4 7 9 7 injection ______________________________________
Example 6
Decomposition of Trichloroethylene in Column with Liquid Agent
Injection Followed by Immediate Air Injection
Columns packed with fine sand were prepared in the same manner as
in Example 1.
Trichloroethylene (TCE) was added to the sand layer of each column
to the concentration of about 10 ppm for experiments.
As a microorganism, strain JM1 (National Institute of Bioscience
and Human-Technology, Agency of Industrial Science and Technology:
FERM BP-5352) was used. It was cultured overnight in an M9 medium
supplemented with 0.1% yeast extract. The number of the
microorganism of the overnight culture, determined by the number of
colonies formed on an agar medium, was 5.times.10.sup.8 cells/ml.
The overnight culture was diluted 500 times with pure water, and
the dilution of 1.times.10.sup.6 cells/ml was injected into the
column as a liquid agent from the bottom. The injection of the
liquid agent was carried out using a peristaltic pump at the
pumping speed for injection of 50 ml/min. The injection of the
liquid agent was stopped when the liquid agent reached 45 cm from
the bottom of the sand layer, and immediately followed by injection
of the air under a pressure of 1 kg/cm.sup.2. Air injection was
continued until the water front in the sand was observed to reach
the top of the sand layer. The water content of the soil
immediately after the liquid injection determined as in Example 1
was about 22-23%. The above-mentioned injection procedure was
carried out on two columns; the fine sand layer was taken out from
one of the columns immediately after the injection of air, while
the other column was tightly sealed with a Teflon seal immediately
after the air injection, and the fine sand was taken out from this
column 5 days later, for trichloroethylene concentration
determination. Sampling of the sand layer was carried out at 9
points, i.e. 5 cm from the bottom of the sand layer to 85 cm from
the bottom with an interval of 10 cm, and the TCE concentration at
each sampling point was measured according to solvent extraction
method using n-hexane. The TCE concentration at each sampling point
is shown in Table 3.
These results show that the microbial cells and water can be
migrated efficiently in the soil and the TCE in the soil can be
decomposed efficiently when the liquid agent injection is followed
by an air injection under pressure through the region of which
water content is 0.6 or more times its saturation water
content.
TABLE 3 ______________________________________ TCE concentration
(ppm) in the column immediately after and 5 days after the
injection of air Distance from the bottom of the sand layer (cm) 5
15 25 35 45 55 65 75 85 ______________________________________
Immediately 6 7 6 8 7 8 9 8 9 after injection 5 days after 0 0 0 0
0 1 0 0 0 injection ______________________________________
Comparative Example 6
Decomposition of TCE in Column with Liquid Agent Injection
Alone
The decomposition of TCE in a column was observed in a way similar
to that used in Example 6 except that air was not injected.
The TCE concentration at each sampling point is shown in Table 4.
These results show that the injection of the liquid agent alone
does not allow the efficient migration of the microbial cells nor
efficient decomposition of TCE in the soil.
TABLE 4 ______________________________________ TCE concentration
(ppm) in the column with liquid agent injection alone Distance from
the bottom of the sand layer (cm) 5 15 25 35 45 55 65 75 85
______________________________________ 5 days after 0 0 0 0 1 2 6 8
7 injection ______________________________________
Example 7
Decomposition of TCE in Pot with Injection of Liquid Agent Followed
by Immediate Air Injection
Pots packed with fine sand were prepared in the similar manner as
that used in Example 3. For experiments, TCE was added to the fine
sand to a concentration of about 10 ppm. As a microorganism, strain
JM1 was used. It was cultured overnight in an M9 medium
supplemented with 0.1% yeast extract. The number of the
microorganism of the overnight culture, determined by the number of
colonies formed on an agar medium, was 5.times.10.sup.8 cells/ml.
The overnight culture was diluted 500 times with pure water, and
the dilution of 1.times.10.sup.6 cells/ml was injected into the pot
as a liquid agent from the bottom. The injection of the liquid
agent was carried out using a peristaltic pump in the center of the
pot, at the depth of 8 cm, and the pumping speed for injection was
200 ml/min. The liquid agent was injected for 5 minutes, then
without delay, about 40 l air was injected under a pressure of 1
kg/cm.sup.2 for 3 minutes through the same injection port. The
water content of the soil immediately after the liquid injection
determined as in Example 3 was about 22-23%.
The above-mentioned injection procedure was carried out in two
pots; the fine sand layer was taken out from one of the pots
immediately after the injection of air, while the other pot was
tightly sealed with a TEFLON brand polytetrafluoroethylene seal
immediately after the air injection, and the fine sand was taken
out from the pot 5 days later, both for trichloroethylene
concentration determination. Sampling of the sand layer was carried
out total at 12 points, i.e. at radii of 6 cm, 9 cm and 12 cm from
the center of the pot, each in the depth of 2 cm, 8 cm, 14 cm and
20 cm from the surface of the soil. The TCE concentration at each
sampling point was measured according to solvent extraction method
using n-hexane. The TCE concentration at each sampling point is
shown in Table 5 and Table 6. These results show that the
microorganisms and water can be migrated efficiently in the soil to
efficiently decompose the TCE in the soil when air was injected
under pressure through the region of which water content is 0.6 or
more times its saturation water content after the injection of the
liquid agent.
TABLE 5 ______________________________________ TCE concentration
(ppm) in the pot immediately after injection of air Distance from
the center of the pot (cm) 6 9 12
______________________________________ Depth(cm) 2 8 9 9 8 6 7 8 14
6 8 9 20 7 8 9 ______________________________________
TABLE 6 ______________________________________ TCE concentration
(ppm) in the pot 5 days after injection of air Distance from the
center of the pot(cm) 6 9 12 ______________________________________
Depth(cm) 2 1 2 2 8 0 0 0 14 0 0 0 20 0 2 9
______________________________________
Comparative Example 7
Decomposition of TCE in Pot with Injection of Liquid Agent
Alone
The decomposition of TCE in a pot was observed 5 days after the
injection of the liquid agent in the same manner as in Example 7
except that air was not injected.
The TCE concentration at each sampling point is shown in Table 7.
These results show that the injection of the liquid agent alone
does not allow the efficient migration of the microbial cells thus
TCE in the soil cannot be efficiently decomposed.
TABLE 7 ______________________________________ TCE concentration
(ppm) in the pot with liquid agent injection alone Distance from
the center of the pot(cm) 6 9 12
______________________________________ Depth(cm) 2 2 8 9 8 0 0 8 14
0 1 2 20 0 2 5 ______________________________________
* * * * *